Journal: Frontiers in Immunology

Article Title: Inhibition of IP3R/Ca2+ Dysregulation Protects Mice From Ventilator-Induced Lung Injury via Endoplasmic Reticulum and Mitochondrial Pathways

doi: 10.3389/fimmu.2021.729094

Figure Lengend Snippet: MV with HTV induces IP3R1 activation in lungs. Immunofluorescence photomicrographs (A) and quantification analysis (B) of IP3R1 in lung tissues of mice with spontaneous breathing (CON group) or mechanical ventilation at high tidal volume (HTV group). Dapi (blue) was used to stain the nuclei. Scale bar: 200μm. An inset picture was employed to show the indicated area at 4X magnification. Western blot (C) and quantification analysis (D) of IP3R1 protein expression in lung extracts. (E) Western blot analysis of the indicated subcellular fractions for IP3R1, calnexin (CNX; ER and MAM marker), FACL-4 (MAM marker) and tubulin (cytosolic marker). (F) Quantification of IP3R1 protein expression in the ER and MAM was performed by normalizing to CNX. Data are expressed as means ± SD (n = 6 animals per group). * P < 0.05 vs . CON group.

Article Snippet: The membranes were blocked with 5% milk in TBST for 1 h at room temperature, incubated with anti-IP3R1 (A4436, ABclonal), anti-FACL-4 (sc-365230, Santa Cruz), anti-Calnexin (AP0635, ABclonal), anti-Tubulin (#2148, CST), anti-GRP78 (sc-166490, Santa Cruz; and/or GB11098, Servicebio), anti-CHOP (sc-7351, Santa Cruz), anti-phospho-IRE1α (ab48187, abcam), anti-IRE1α (ab37073, abcam), anti-TRAF2 (#4724, CST), anti-XBP-1s (#40435, CST), anti-phospho-PERK (#3179, CST), anti- PERK (#5683, CST), anti- phospho- eIF2α (AP0635, ABclonal), anti- eIF2α(A0764, ABclonal), anti-ATF6 (ab37149, abcam), anti-IκBα (#4814s, CST), anti-p-NF-κB p65 (Ser536, #3033s, CST), anti-NF-κB p65 (#8242s, CST), anti-Lamin B (sc-374015, Santa Cruz), anti- NLRP3 (#13158, CST), anti-caspase-1 (A0964, ABclonal), anti-ASC (67824S, CST), anti-β-actin (#4970, CST).

Techniques: Activation Assay, Immunofluorescence, Staining, Western Blot, Expressing, Marker

Journal: Ecotoxicology and environmental safety

Article Title: Thiram-induced ER stress promotes mitochondrial calcium signaling and NLRP3 inflammasome activation in a tissue specific manner.

doi: 10.1016/j.ecoenv.2025.118026

Figure Lengend Snippet: Fig. 4. Mitochondrial outer membranes associated with VDAC1s hyperactivation triggered by thiram-induced ER stress. (A, B) The expression of the VDAC1 gene in the liver and GP of thiram-supplemented chickens vs control ones (n = 3). (C, D) Bones were subjected to dual-energy X-ray for analysis of bone mineral density (BMD) and bone mineral concentration (BMC) and showing significantly decreased BMC and BMD in thiram supplemented groups. (E, F) The western blot bands of VDAC1 protein and their quantification from different groups (n = 3) in liver tissues. (G, H) ImageJ quantification and western blot bands of tibial GP at days 14th and 28thhere both of the bands indicate VDACs expression possibility due to cross-reactivity with other VDAC’s isoforms in chicken tissues. Bars indicates the means ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Antibodies against GRP78/BiP (WL03157; Shenyang Wanlei Biotechnology Co., Ltd. China), IP3R1 (A4436; ABclonal Technology Co., Ltd, Wuhan, China), VDAC1 (4661 T; Cell signaling Technology, Boston, USA) and NLRP3 (WL02635; Shenyang Wanlei Biotechnology Co., Ltd. China).

Techniques: Expressing, Control, Concentration Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Inhibition of IP3R/Ca2+ Dysregulation Protects Mice From Ventilator-Induced Lung Injury via Endoplasmic Reticulum and Mitochondrial Pathways

doi: 10.3389/fimmu.2021.729094

Figure Lengend Snippet: 2-APB inhibits ER stress in HTV-treated mice. (A) Immunofluorescence photomicrographs of GRP78 (green) and CHOP (red) in lung tissues of group CON, HTV, and HTV+2-APB mice. Dapi (blue) was used to stain the nuclei. Scale bar: 200μm. An inset picture was employed to show the indicated area at 4X magnification. (B, C) Graphic presentations of fluorescence mean densities of GRP78 and CHOP. (D) Levels of GRP78, CHOP, p-IRE1α, t-IRE1α, TRAF2, XBP-1s, p-PERK, t-PERK, p-eIF2α, t-eIF2α, t-ATF6, β-actin proteins by Western blot. (E) Relative protein expression of GRP78, CHOP, TRAF2, XBP-1s and t-ATF6 relative to β-actin. (F) The relative ratio of p-IRE1α protein was presented to t-IRE1α. (G) The relative ratio of p-PERK protein was presented to t-PERK. (H) The relative ratio of p-eIF2α protein was presented to t-eIF2α. Data are expressed as means ± SD (n = 6 per group). * P < 0.05 vs . CON group. # P < 0.05 vs . HTV group.

Article Snippet: The membranes were blocked with 5% milk in TBST for 1 h at room temperature, incubated with anti-IP3R1 (A4436, ABclonal), anti-FACL-4 (sc-365230, Santa Cruz), anti-Calnexin (AP0635, ABclonal), anti-Tubulin (#2148, CST), anti-GRP78 (sc-166490, Santa Cruz; and/or GB11098, Servicebio), anti-CHOP (sc-7351, Santa Cruz), anti-phospho-IRE1α (ab48187, abcam), anti-IRE1α (ab37073, abcam), anti-TRAF2 (#4724, CST), anti-XBP-1s (#40435, CST), anti-phospho-PERK (#3179, CST), anti- PERK (#5683, CST), anti- phospho- eIF2α (AP0635, ABclonal), anti- eIF2α(A0764, ABclonal), anti-ATF6 (ab37149, abcam), anti-IκBα (#4814s, CST), anti-p-NF-κB p65 (Ser536, #3033s, CST), anti-NF-κB p65 (#8242s, CST), anti-Lamin B (sc-374015, Santa Cruz), anti- NLRP3 (#13158, CST), anti-caspase-1 (A0964, ABclonal), anti-ASC (67824S, CST), anti-β-actin (#4970, CST).

Techniques: Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: Inhibition of IP3R/Ca2+ Dysregulation Protects Mice From Ventilator-Induced Lung Injury via Endoplasmic Reticulum and Mitochondrial Pathways

doi: 10.3389/fimmu.2021.729094

Figure Lengend Snippet: MV with HTV induces IP3R1 activation in lungs. Immunofluorescence photomicrographs (A) and quantification analysis (B) of IP3R1 in lung tissues of mice with spontaneous breathing (CON group) or mechanical ventilation at high tidal volume (HTV group). Dapi (blue) was used to stain the nuclei. Scale bar: 200μm. An inset picture was employed to show the indicated area at 4X magnification. Western blot (C) and quantification analysis (D) of IP3R1 protein expression in lung extracts. (E) Western blot analysis of the indicated subcellular fractions for IP3R1, calnexin (CNX; ER and MAM marker), FACL-4 (MAM marker) and tubulin (cytosolic marker). (F) Quantification of IP3R1 protein expression in the ER and MAM was performed by normalizing to CNX. Data are expressed as means ± SD (n = 6 animals per group). * P < 0.05 vs . CON group.

Article Snippet: The membranes were blocked with 5% milk in TBST for 1 h at room temperature, incubated with anti-IP3R1 (A4436, ABclonal), anti-FACL-4 (sc-365230, Santa Cruz), anti-Calnexin (AP0635, ABclonal), anti-Tubulin (#2148, CST), anti-GRP78 (sc-166490, Santa Cruz; and/or GB11098, Servicebio), anti-CHOP (sc-7351, Santa Cruz), anti-phospho-IRE1α (ab48187, abcam), anti-IRE1α (ab37073, abcam), anti-TRAF2 (#4724, CST), anti-XBP-1s (#40435, CST), anti-phospho-PERK (#3179, CST), anti- PERK (#5683, CST), anti- phospho- eIF2α (AP0635, ABclonal), anti- eIF2α(A0764, ABclonal), anti-ATF6 (ab37149, abcam), anti-IκBα (#4814s, CST), anti-p-NF-κB p65 (Ser536, #3033s, CST), anti-NF-κB p65 (#8242s, CST), anti-Lamin B (sc-374015, Santa Cruz), anti- NLRP3 (#13158, CST), anti-caspase-1 (A0964, ABclonal), anti-ASC (67824S, CST), anti-β-actin (#4970, CST).

Techniques: Activation Assay, Immunofluorescence, Staining, Western Blot, Expressing, Marker

Journal: MedComm

Article Title: cZFP609 Tethering BiP Alleviates Cartilage Degradation in Osteoarthritis via Remedying Aberrant ER‐Mitochondrial Contacts

doi: 10.1002/mco2.70405

Figure Lengend Snippet: Osteoarthritis is ameliorated in the OA model of sm Sirt1 ‐Tg mice. (A–D) WT and sm Sirt1 ‐Tg mice were induced by DMM surgery ( n = 6). Safranin‐O/fast green staining (A) and scoring of OA parameters, including OARSI grade (B), chondrocyte number (C), and cartilage thickness (D). Scale bar = 100 µm. (E, F) Immunohistochemical staining for p53, MMP13, Sox9, and Col2ɑ proteins in the cartilage tissue (E) and corresponding quantitative analysis (F). Scale bar = 50 µm. (G, H) Western blot analysis of protein levels of p53, MMP13, Col2ɑ, Sox9, and Sirt1 in the cartilage tissue (G) and corresponding quantitative analysis (H). (I, J) PWT (I) and PWL (J) were used to evaluate mechanical pain sensitivity and hyperalgesia. Data and images represent at least three independent experiments. Statistical analyses, paired t ‐tests, and Tukey's multiple comparisons tests. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus the corresponding control.

Article Snippet: The membranes were blocked in 5% milk for 1 h at room temperature and then incubated with primary antibodies against MMP13 (1:1000, 18165‐1‐AP, Proteintech), Col2ɑ (1:1000, 28459‐1‐AP, Proteintech), Sox9 (1:200, sc‐166505, Santa Cruz), p53 (1:1000, 10442‐1‐AP, Proteintech), p16 (1:1000, A0262, ABclonal), p21(1:1000, HA500005 , HUABIO), Sirt1 (1:500, A11267, ABclonal), IL‐1β (1:500, 16806‐1‐AP, Proteintech), BiP (1:1000, 11587‐1‐AP, Proteintech), IRE1α (1:1000, 27528‐1‐AP, Proteintech), IRE1α‐p (1:1000, AP0878, ABclonal), XBP‐1S (1:1000, 24868‐1‐AP, Proteintech), ATF4 (1:1000, 60035‐1‐Ig, Proteintech), IP3R (1:1000, A4436, ABclonal), TFRC (1:1000, A5865, ABclonal), GPX4 (1:1000, A11243, ABclonal), FTH (1:1000, 11682‐1‐AP, Proteintech), ACSL4 (1:1000, 22401‐1‐AP, Proteintech), COX2 (1:1000, ET1610‐23, HUABIO), and GAPDH (1:2000, AC036, ABclonal) at 4°C overnight.

Techniques: Staining, Immunohistochemical staining, Western Blot, Control

Journal: MedComm

Article Title: cZFP609 Tethering BiP Alleviates Cartilage Degradation in Osteoarthritis via Remedying Aberrant ER‐Mitochondrial Contacts

doi: 10.1002/mco2.70405

Figure Lengend Snippet: Overexpression of cZFP609 protects chondrocytes from inflammation and degeneration. (A–E) RT‐qPCR of cZFP609 expression. (A) In the articular cartilage (left) and plasma (right) of WT and sm Sirt1 ‐Tg mice ( n = 6). (B) The plasma level of cZFP609 in OA patients and normal control subjects ( n = 20). (C) In the VSMCs of WT and sm Sirt1 ‐Tg mice. (D) In WT and sm Sirt1 ‐Tg VSMC conditional media (CM). (E) Chondrocytes were incubated with WT and sm Sirt1 ‐Tg VSMCs CM for 24 h. (F–H) Western blot analysis of p16, p21, p53, IL‐1β, and MMP13 in chondrocytes. (F) Incubated with WT and sm Sirt1 ‐Tg VSMCs CM after treatment with TNFα (20 ng/mL). (G) Incubated with CM from the cZFP609 siRNA‐transfected sm Sirt1 ‐Tg VSMCs. (H) Transfected with vector or cZFP609 plasmid DNA for 12 h and then treated with TNFα (20 ng/mL) for 48 h. (I) SA‐β‐gal staining and levels. Scale bar = 200 µm. Data and images represent at least three independent experiments by paired t ‐test and Sidak's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus the corresponding control.

Article Snippet: The membranes were blocked in 5% milk for 1 h at room temperature and then incubated with primary antibodies against MMP13 (1:1000, 18165‐1‐AP, Proteintech), Col2ɑ (1:1000, 28459‐1‐AP, Proteintech), Sox9 (1:200, sc‐166505, Santa Cruz), p53 (1:1000, 10442‐1‐AP, Proteintech), p16 (1:1000, A0262, ABclonal), p21(1:1000, HA500005 , HUABIO), Sirt1 (1:500, A11267, ABclonal), IL‐1β (1:500, 16806‐1‐AP, Proteintech), BiP (1:1000, 11587‐1‐AP, Proteintech), IRE1α (1:1000, 27528‐1‐AP, Proteintech), IRE1α‐p (1:1000, AP0878, ABclonal), XBP‐1S (1:1000, 24868‐1‐AP, Proteintech), ATF4 (1:1000, 60035‐1‐Ig, Proteintech), IP3R (1:1000, A4436, ABclonal), TFRC (1:1000, A5865, ABclonal), GPX4 (1:1000, A11243, ABclonal), FTH (1:1000, 11682‐1‐AP, Proteintech), ACSL4 (1:1000, 22401‐1‐AP, Proteintech), COX2 (1:1000, ET1610‐23, HUABIO), and GAPDH (1:2000, AC036, ABclonal) at 4°C overnight.

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Clinical Proteomics, Control, Incubation, Western Blot, Transfection, Plasmid Preparation, Staining

Journal: MedComm

Article Title: cZFP609 Tethering BiP Alleviates Cartilage Degradation in Osteoarthritis via Remedying Aberrant ER‐Mitochondrial Contacts

doi: 10.1002/mco2.70405

Figure Lengend Snippet: cZFP609 suppresses ferroptosis of chondrocytes. (A) Representative images of BODIPY (581/591) C11 staining in chondrocytes treated with the indicated conditions. The relative fluorescence intensity of R‐Bodipy/Ox‐Bodipy was quantified using Fiji images. Scale bar = 20 µm. (B) Representative images and relative quantification of FerroOrange staining. Scale bar = 40 µm. (C, D) MDA (C) and GSH (D) levels were detected in chondrocytes. (E) Western blot analysis of TFRC, ACSL4, COX2, FTH, and GPX4 proteins in chondrocytes. (F) Representative images from flow cytometry in chondrocytes. The total percentage of apoptosis (red square) is equal to the percentage of early apoptosis (Q2, Annexin V + 7‐AAD + ) plus the percentage of late apoptosis (Q3, Annexin V + 7‐AAD + ). (G) The cell viability of the chondrocytes was determined by CCK‐8 analysis. (H) Representative images and relative quantification of FerroOrange staining in chondrocytes treated with the indicated conditions. Scale bar = 40 µm. (I) Representative images of BODIPY (581/591) C11 staining and relative quantification. Scale bar = 20 µm. Data and images represent at least three independent experiments by Sidak's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus the corresponding control.

Article Snippet: The membranes were blocked in 5% milk for 1 h at room temperature and then incubated with primary antibodies against MMP13 (1:1000, 18165‐1‐AP, Proteintech), Col2ɑ (1:1000, 28459‐1‐AP, Proteintech), Sox9 (1:200, sc‐166505, Santa Cruz), p53 (1:1000, 10442‐1‐AP, Proteintech), p16 (1:1000, A0262, ABclonal), p21(1:1000, HA500005 , HUABIO), Sirt1 (1:500, A11267, ABclonal), IL‐1β (1:500, 16806‐1‐AP, Proteintech), BiP (1:1000, 11587‐1‐AP, Proteintech), IRE1α (1:1000, 27528‐1‐AP, Proteintech), IRE1α‐p (1:1000, AP0878, ABclonal), XBP‐1S (1:1000, 24868‐1‐AP, Proteintech), ATF4 (1:1000, 60035‐1‐Ig, Proteintech), IP3R (1:1000, A4436, ABclonal), TFRC (1:1000, A5865, ABclonal), GPX4 (1:1000, A11243, ABclonal), FTH (1:1000, 11682‐1‐AP, Proteintech), ACSL4 (1:1000, 22401‐1‐AP, Proteintech), COX2 (1:1000, ET1610‐23, HUABIO), and GAPDH (1:2000, AC036, ABclonal) at 4°C overnight.

Techniques: Staining, Fluorescence, Quantitative Proteomics, Western Blot, Flow Cytometry, CCK-8 Assay, Control

Journal: MedComm

Article Title: cZFP609 Tethering BiP Alleviates Cartilage Degradation in Osteoarthritis via Remedying Aberrant ER‐Mitochondrial Contacts

doi: 10.1002/mco2.70405

Figure Lengend Snippet: Intra‐articular injection of cZFP609 plasmid alleviates OA progression. (A) Schematic of the experimental timeline receiving sham or DMM surgery and intra‐articular cZFP609 plasmid DNA (pDNA) injection in wild‐type mice ( n = 6). (B) The efficiency of cZFP609 was determined by RT‐qPCR in cartilage tissue at sacrifice. (C–F) Safranin‐O/fast green staining (C) and scoring of OA parameters, including OARSI grade (D), chondrocyte number (E), and cartilage thickness (F). Scale bar = 100 µm ( n = 6). (G, H) PWT (G) and PWL (H) were used to evaluate mechanical pain sensitivity and hyperalgesia. (I, J) Immunohistochemical staining of GPX4 and ACSL4 proteins in the cartilage tissue (I) and quantitative analysis (J). Scale bar = 50 µm. (K, L) Western blot (K) and quantification analysis (L) of the protein levels of GPX4, ACSL4, and COX2 in the cartilage tissue. Data and images represent at least three independent experiments. Statistical analyses, paired t ‐tests, and Tukey's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 versus the corresponding control.

Article Snippet: The membranes were blocked in 5% milk for 1 h at room temperature and then incubated with primary antibodies against MMP13 (1:1000, 18165‐1‐AP, Proteintech), Col2ɑ (1:1000, 28459‐1‐AP, Proteintech), Sox9 (1:200, sc‐166505, Santa Cruz), p53 (1:1000, 10442‐1‐AP, Proteintech), p16 (1:1000, A0262, ABclonal), p21(1:1000, HA500005 , HUABIO), Sirt1 (1:500, A11267, ABclonal), IL‐1β (1:500, 16806‐1‐AP, Proteintech), BiP (1:1000, 11587‐1‐AP, Proteintech), IRE1α (1:1000, 27528‐1‐AP, Proteintech), IRE1α‐p (1:1000, AP0878, ABclonal), XBP‐1S (1:1000, 24868‐1‐AP, Proteintech), ATF4 (1:1000, 60035‐1‐Ig, Proteintech), IP3R (1:1000, A4436, ABclonal), TFRC (1:1000, A5865, ABclonal), GPX4 (1:1000, A11243, ABclonal), FTH (1:1000, 11682‐1‐AP, Proteintech), ACSL4 (1:1000, 22401‐1‐AP, Proteintech), COX2 (1:1000, ET1610‐23, HUABIO), and GAPDH (1:2000, AC036, ABclonal) at 4°C overnight.

Techniques: Injection, Plasmid Preparation, Quantitative RT-PCR, Staining, Immunohistochemical staining, Western Blot, Control